張瀞仁臺灣大學：生化科學研究所陳柏洲Chen, Bo-ChouBo-ChouChen2007-11-262018-07-062007-11-262018-07-062007http://ntur.lib.ntu.edu.tw//handle/246246/52732DDX1 is a DEAD box-containing RNA helicase. RNA helicases have been found involved in all aspects of RNA metabolism, including transcription, pre-mRNA processing, RNA export and translation. However, the functional role of DDX1 is unclear.We found that DDX1 was a homopolymeric poly(A) RNA binding protein. We examine the subcellular distribution of DDX1 to identify possible biological functions of DDX1. DDX1 is predominantly present in the nucleus. It could be exported to the cytoplasm in response to arsenite treatment, a oxidative stress stimulus. When tristetraprolin(TTP) was overexpressed to form granular foci in the cytoplasm, immunofluorescence staining followed by confocal microscopy observation showed that DDX1 partially colocalized with TTP. In this condition, DDX1 could colocalize with HuR and TIA-1, which were markers of stress granules(SGs) formed under stress environment. Since TTP was a component of processing bodies(PBs) and SGs, our results indicated DDX1 was localized in SGs. Moreover, we observed that DDX1 could inhibit the translational activity in the stress environment. We suggest that DDX1 could relocalize into SGs as stress stimulation and there were interactions between DDX1 and TTP、 HuR, and decrease the translational activity of RNAs.目錄 ---------------------------------------------------------------------------------------- 2 中文摘要 ---------------------------------------------------------------------------------- 4 英文摘要 ---------------------------------------------------------------------------------- 5 縮寫表 ------------------------------------------------------------------------------------- 6 1. 緒論 ------------------------------------------------------------------------------------- 7 2. 材料與方法 2.1 質體構築 ------------------------------------------------------------------------- 12 2.2 細胞培養 ------------------------------------------------------------------------- 12 2.3 去氧核醣核酸轉染以及化學藥劑的處理 ---------------------------------- 13 2.4 細胞萃取液的配製 ------------------------------------------------------------- 13 2.5 細胞核與細胞質的分離 ------------------------------------------------------- 13 2.6 SDS聚丙醯胺膠體電泳 ------------------------------------------------------- 14 2.7 西方墨點法 ---------------------------------------------------------------------- 15 2.8 抗體使用 ------------------------------------------------------------------------- 16 2.9 核醣核酸結合能力分析 ------------------------------------------------------- 16 2.10 免疫沈澱分析 ------------------------------------------------------------------- 16 2.11 多核醣體分析 ------------------------------------------------------------------- 17 2.12 細胞免疫染色 ------------------------------------------------------------------- 17 2.13 luciferase以及β-galactosidase 分析 -------------------------------------- 18 2.14 銀染 ------------------------------------------------------------------------------- 18 2.14 氨基酸序列分析 ---------------------------------------------------------------- 18 3. 結果 3.1 DDX1具有與 poly(A) 核醣核酸結合的能力 ---------------------------- 20 3.2 DDX1不會與PABP結合也不會與多核醣體互相作用 ------------------ 20 3.3 以砷處理細胞時，DDX1會改變在細胞中的分佈情形 ----------------- 21 3.4 在逆境環境下，DDX1會位於逆境小體中 -------------------------------- 22 3.5 DDX1分別與TTP、HuR有相互結合 -------------------------------------- 24 3.6 DDX1會抑制轉譯反應的進行 ------------------------------------------------ 24 3.7 與DDX1結合蛋白之鑑定分析 ------------------------------------------------- 25 4. 討論 --------------------------------------------------------------------------------------- 27 5. 參考資料 --------------------------------------------------------------------------------- 33 6. 圖表 --------------------------------------------------------------------------------------- 42 7. 補充資料 --------------------------------------------------------------------------------- 55en-US核醣核酸解螺逆境小體DDX1stress granuleother